One of the major forms of communication between neurons is that which occurs at chemical synapses. The control of the efficiency of synaptic communication is a crucial means by which the nervous system directs information flow in the brain. Understanding the detailed physiology of synaptic terminals in the central nervous system (CNS) will thus be critical to determining the nature of this control in both normal and diseased states of brain function. Our long term objectives are to determine the sub-cellular processes and their molecular substrates, that control the efficiency of synaptic transmission in the CNS. Or approach is to use optical tracer approaches that allow one to dissect out various cell biological parameters that directly impact presynaptic function. Our previous work demonstrates the usefulness of fluorescent tracers such as FM 1-43 as well as the newly developed pH-based optical sensors, synapto-pHluorins for use in hippocampal neurons in culture for providing robust quantitative measures of exocytosis, endocytosis, vesicle repriming, recycling and non-recycling vesicle pool sizes. We propose four specific aims to examine the biophysical and molecular basis of presynaptic function. 1) Examine the role of the abundant synaptic vesicle protein synaptotagmin I in all aspects of vesicle recycling using opto-physiological methods in neurons derived from synaptotagmin I-deficient mice and dissect the role of the calcium-binding domains in controlling specific vesicle cycling steps. 2) Determine how known intracellular second messengers as well as members of the synapsin family control the recycling and non-recycling vesicle pool size at individual synaptic terminals. 3) Characterize detailed parameters of vesicle recycling in excitatory versus inhibitory synaptic terminals in hippocampal neurons. 4) Determine how known exogenous modulators of synaptic transmission (glutatamate, neurotrophins) control vesicle pool mobilization at individual hippocampal synaptic terminals.